Multi-Way Sorter System and Method

ABSTRACT

Provided herein are improved multi-way cell sorter systems and methods. For example, provided are systems and methods for the collection of cells that are sorted in multiple directions. The systems and methods allow for the construction of a multi-way sorter (e.g., a ten-way sorter in the space that currently only allows four-way sorting). In addition the device may actively sense the arrival of drops (with cells of interest) at a sample tube, and trigger an alarm when the drops&#39; arrivals deviate from an expected pattern.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e) this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 61/480,867filed Apr. 29, 2011; the disclosure of which application is hereinincorporated by reference.

INTRODUCTION

The present invention relates to flow cytometers and instruments forhigh speed identification and sorting of particles, such as cells.

Flow cytometry is a valuable method for the analysis and isolation ofbiological particles such as cells and constituent molecules. As such ithas a wide range of diagnostic and therapeutic applications. The methodutilizes a fluid stream to linearly segregate particles such that theycan pass, single file, through a detection apparatus. Individual cellscan be distinguished according to their location in the fluid stream andthe presence of detectable markers. Thus, a flow cytometer can be usedto produce a diagnostic profile of a population of biological particles.

Isolation of biological particles has been achieved by adding a sortingor collection capability to flow cytometers. Particles in a segregatedstream, detected as having one or more desired characteristics, areindividually isolated from the sample stream by mechanical or electricalremoval. A common flow sorting technique utilizes drop sorting in whicha fluid stream containing linearly segregated particles is broken intodrops and the drops containing particles of interest are electricallycharged and deflected into a collection tube by passage through anelectric field.

Typically, the linearly segregated particles in the stream arecharacterized as they pass through an observation point situated justbelow the nozzle tip. Once a particle is identified as meeting one ormore desired criteria, the time at which it will reach the dropbreak-off point and break from the stream in a drop can be predicted.Ideally, a brief charge is applied to the fluid stream just before thedrop containing the selected particle breaks from the stream and thengrounded immediately after the drop breaks off. The drop to be sortedmaintains an electrical charge as it breaks off from the fluid stream,and all other drops are left uncharged. The charged drop is deflectedsideways from the downward trajectory of the other drops by anelectrical field and collected in a sample tube. The uncharged dropsfall directly into a drain.

Droplet sorters deflect drops that contain cells of interest by applyinga charge (typically 50-150 volts) to the drops. The drops are deflectedby an electrostatic field and, depending on the charge, follow differenttrajectories so that that arrive at a collection tube. The cells can bedistributed over multiple groups by applying different charges, eachdiscrete charge guiding a drop to a particular tube. Originally cellpopulations were divided in to two groups: a left and a right sort.lately the number of sort directions (groups) has been increased to 4(most sorters) or even 6. An example flow cytometer system havingmultiple collection tubes is provided in U.S. Pat. No. 5,483,469, whichis incorporated herein by reference in its entirety.

The number of sort directions is currently limited by the number ofcollection tubes that can be placed on the axis along which the dropsare distributed. The typical drop spread in one direction is about 22mm. The tube centers are 14 mm apart so that with difficulty threegroups can be sorted.

The tube size is not the only factor in limiting the number of sortdirections. It is also difficult to place sorted drops in closeproximity because the drops carry a charge. As such, charged drops willrepel each other. As the drops accumulate into a bigger volume thecharge grows disproportionally faster. Due to the accumulation ofcharge, the sorted cell volume tends to want to break into smallerdrops. These drops repel and push away each other causing drops to jumparound erratically, sometimes even jumping into adjacent tubes withdrops of opposite charge, thus severely affecting the purity of a sort.

SUMMARY

Provided herein are improved multi-way cell sorter systems and methods.For example, provided are systems and methods for the collection ofcells that are sorted in multiple directions. The systems and methodsallow for the construction of a multi-way sorter (e.g., a ten-way sorterin the space that currently only allows four-way sorting). In additionthe device may actively sense the arrival of drops (with cells ofinterest) at a sample tube, and trigger an alarm when the drops'arrivals deviate from an expected pattern. Such a signal could be usedto move the collection tubes into a safe position preventingcontamination of the sorted samples. This device is desirable forclinical sorting. The device is also suitable as a disposable unit.

The systems and methods presented provide a cure for the dropaccumulation problem, and allow deflected drop streams to be placed incloser proximity without affecting stream stability.

Exemplary systems and methods presented include a sorted drop receptacle(or carrier) that consists of a row of sort tubes (or deflection tubes)that are open at both sides. The deflected drops are accordingly aimedat the inside of a sort tube. Each sort tube is individually grounded sothat the deflection charge on the drops is harmlessly deflected toground. Thus the drops no longer carry a charge and can grow into largerdrops that slide down the sort tube. Each sort tube is inserted in anopening of a sample collection tube. Since the sort tubes can be bent,the collection tubes no longer need to be placed along a line, but canbe configured in a staggered pattern. As such, a collection area thatonce held three tubes in a straight line, can now accommodate five tubesin a staggered configuration. Since the sort tubes can be narrower thanthe collection tubes, the tops of the five sort tubes can be placedalong a 20 mm line, with the bottom of the tubes leading to fivestaggered collection tubes place in a 20×30 mm area.

In one embodiment, an arrangement of ten collection tubes with ten sorttubes can be fitted on the area of a standard microscope slide. Aplastic carrier with the dimensions of a microscope slide may hold aboutten thin walled stainless steel deflection tubes. The ten deflectiontubes lead to ten plastic collection tubes that are attached to thebottom of the plastic carrier. The whole unit may be made available asone sterile and disposable unit.

The top of the sort tubes may also be closed off when appropriate. Forexample, the system can monitor the arrival of drops in the sortfractions by measuring the charge that is deposited in the individualmetal sort tubes. If the metal sort tubes are connected to ground via acurrent-to-voltage converter, with a variable integration time, thevoltage measured at each sort tube reflects the number of drops that aredeposited in each tube per-unit-time. A computer program can beconfigured to predict the expected voltage generated for each sortdirection. If the measured voltage differs from the expected voltage,the computer can instruct a stepper motor to cover the sort tubes, ormove the collection tray into a safe, covered (or otherwise deactivated)position, preventing contamination of the sorted fractions with unwantedcells.

The carrier presented may be used as a multi-way (e.g. ten-way) sorterin all current influx sorters, without the need for any changes to theinflux design. The possibility to continuously monitor the number ofcells that are deposited in each tube also provides a safeguard thatcurrently is not available on cell sorters. This control device shouldbe very desirable for sorts that require a high degree of confidence inthe purity of the cell fractions.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated herein, form part ofthe specification. Together with this written description, the drawingsfurther serve to explain the principles of, and to enable a personskilled in the relevant art(s), to make and use systems and methods inaccordance with the present invention.

FIG. 1 is a perspective view of a portion of a multi-way cell sortercarrier, in accordance with one embodiment presented herein.

FIG. 2 is a side view of a portion of the multi-way cell sorter carrierof FIG. 1.

FIG. 3 is a front view of a portion of the multi-way cell sorter carrierof FIG. 1.

FIG. 4 is a top view of a portion of the multi-way cell sorter carrierof FIG. 1.

DETAILED DESCRIPTION

The following detailed description of the figures refers to theaccompanying drawings that illustrate an exemplary embodiment of acarrier for a cell sorter system. Other embodiments are possible.Modifications may be made to the embodiments described herein withoutdeparting from the spirit and scope of the present invention. Therefore,the following detailed description is not meant to be limiting.

FIG. 1 is a perspective view of a portion of a multi-way cell sortercarrier 100, in accordance with one embodiment presented herein. FIG. 2is a side view of a portion of multi-way cell sorter carrier 100. FIG. 3is a front view of a portion of multi-way cell sorter carrier 100. FIG.4 is a top view of a portion of multi-way cell sorter carrier 100.Carrier 100 may be incorporated into a cell sorter system. An exemplarycell sorter system is provided in U.S. Pat. No. 5,483,469, which isincorporated herein by reference in its entirety.

In the embodiment shown, carrier 100 includes a plurality of deflection(or sort) tubes 102 and corresponding collection tubes 104. Eachdeflection tube 102 is hollow, with openings at both ends. Openings 106of the deflection tubes 102 are aligned collinearly with one another,along an axis of the deflection. As such, when carrier 100 isincorporated into a cell sorter system, openings 106 of deflection tubes102 receive deflected charged droplets.

A second opening of each deflection tube 102 is positioned with respectto a corresponding opening in a collection tube 104. As such, chargeddroplets received within deflection tubes 102 slide into collectiontubes 104. Collection tubes 104 are preferably positioned in a staggeredarrangement in order to maximize the spacing and number of collectiontubes used in carrier 100. To facilitate positioning of deflection tubes102 and collection tubes 104, and/or to secure the connection betweendeflection tubes 102 and collection tubes 104, a carrier plate 108 isused. As shown, only five deflection tubes 102 and five collection tubes104 are presented. However, in one embodiment, up to ten or moredeflection tubes and collection tubes may be used.

In one embodiment, each deflection tube 102 is formed of an electricallyconductive material (e.g., a metal, such as, stainless steel). Eachcollection tube 104 may be formed of a lightweight plastic material.Carrier plate 108 may be formed of a plastic material.

In another embodiment, each the deflection tube 102 is further coupledto a current-to-voltage converter (CVC) (not shown), which isappropriately grounded. The grounding of the CVC allows for the removalof the charge of the drops received in the deflection tube 102. Further,CVC allows for the measurement of current at deflection tube 102, whichis a function of the charged droplet arriving at deflection tube 102. Assuch, the arrival of drops at carrier 100 can be monitored by measuringthe charge that is deposited in the individual deflection tubes 102. Ifthe deflection tubes 102 are connected to ground via CVC, with avariable integration time, the voltage measured at each deflection tubereflects the number of drops that are deposited in each tubeper-unit-time. A computer program can be configured to predict theexpected voltage generated for each sort direction. If the measuredvoltage differs from the expected voltage, the computer can instruct astepper motor to cover the carrier, or move the carrier into a safe,covered, and/or deactivated position, preventing contamination of thecarrier with unwanted cells.

In yet another embodiment, each deflection tube 102 may include twosegmented pieces 102 a, 102 b. The segmentation of the deflection tubemay be formed by a “bend” in the tube. Since the deflection tubes can bebent, the collection tubes no longer need to be placed along a line, butcan be configured in a staggered pattern. As such, a collection areathat once held three tubes in a straight line, can now accommodate fivetubes in a staggered configuration. Since the deflection tubes can benarrower than the collection tubes, the tops of the five deflectiontubes can be placed along a 20 mm line, with the bottom of the tubesleading to five staggered collection tubes placed in a 20×30 mm area.

In still another embodiment, there is provided a carrier for receivingcells sorted from a cell sorter system. The carrier includes a pluralityof hollow deflection tubes and a plurality of corresponding collectiontubes. Each deflection tube includes a first opening at a first end anda second opening at a second end. The first opening of each deflectiontube is aligned with the first opening of an adjacent deflection tubesuch that the first openings are all collinear. The second openings ofthe plurality of deflection tubes are staggered with respect to oneanother. Each collection tube includes an opening positioned to receivea cell passed through and exiting a corresponding deflection tube. Inone embodiment, each deflection tube and/or each collection tube iselectrically insulated. In one embodiment, the carrier further includesa current detection circuit coupled to at least one deflection tube, ora current detection circuit coupled to each deflection tube. The currentdetection circuit may further include a current-to-voltage converter todetect the presence of a charged drop entering the deflection tube,and/or an integrator to detect a number of drops per-unit-time enteringthe deflection tube. Each deflection tube may be formed of a metallicmaterial. Each collection tube may be formed of a plastic material. Inone embodiment, the carrier includes ten or more deflection tubes andten or more corresponding collection tubes.

Further, there is provided a method for configuring a carrier forreceiving cells sorted by a cell sorter system. The method includesaligning a plurality of hollow deflection tubes such that a first openend of each deflection tube is positioned collinearly with a first openend of an adjacent deflection tube. A second open end of each deflectiontube is positioned staggered with respect to a second open end of anadjacent deflection tube. The method further includes positioning aplurality of corresponding collection tubes such that an opening foreach collection tube is positioned to receive a cell passed through andexiting a corresponding deflection tube. Each deflection tube may beelectrically insulated. Each collection tube may be electricallyinsulated. The method may further include coupling a current detectioncircuit to at least one deflection tube, or coupling a current detectioncircuit to each deflection tube. The current detection circuit mayinclude: a current-to-voltage converter to detect the presence of acharged drop entering the deflection tube; and/or an integrator todetect a number of drops per-unit-time entering the deflection tube.Each deflection tube may be formed of a metallic material. Eachcollection tube may be formed of a plastic material. The carrier may beconfigured to include ten or more deflection tubes and ten or morecorresponding collection tubes.

Conclusion

The foregoing description of the invention has been presented forpurposes of illustration and description. It is not intended to beexhaustive or to limit the invention to the precise form disclosed.Other modifications and variations may be possible in light of the aboveteachings. The embodiments were chosen and described in order to bestexplain the principles of the invention and its practical application,and to thereby enable others skilled in the art to best utilize theinvention in various embodiments and various modifications as are suitedto the particular use contemplated. It is intended that the appendedclaims be construed to include other alternative embodiments of theinvention; including equivalent structures, components, methods, andmeans.

It is to be appreciated that the Detailed Description section, and notthe Summary and Abstract sections, is intended to be used to interpretthe claims. The Summary and Abstract sections may set forth one or more,but not all exemplary embodiments of the present invention ascontemplated by the inventor(s), and thus, are not intended to limit thepresent invention and the appended claims in any way.

It is to be understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodiments arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace operableprocesses and/or devices/systems/kits. In addition, all sub-combinationslisted in the embodiments describing such variables are alsospecifically embraced by the present invention and are disclosed hereinjust as if each and every such sub-combination of chemical groups wasindividually and explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

1.-23. (canceled)
 24. An article configured to receive cells sorted froma cell sorter system, the article comprising: a plurality of hollowdeflection tubes, wherein each deflection tube has a first opening at afirst end and a second opening at a second end; a plurality ofcollection tubes corresponding to the plurality of deflection tubes,wherein each collection tube includes an opening positioned to receive acell passed through and exiting a corresponding deflection tube; and acurrent detection circuit coupled to at least one of the plurality ofhollow deflection tubes.
 25. The article according to claim 24, whereineach deflection tube is electrically insulated.
 26. The articleaccording to claim 24, wherein each collection tube is electricallyinsulated.
 27. The article according to claim 24, further comprising acurrent detection circuit coupled to each deflection tube.
 28. Thearticle according to claim 24, wherein the current detection circuitcomprises a current-to-voltage converter to detect the presence of acharged drop entering the deflection tube.
 29. The article according toclaim 24, wherein the current detection circuit comprises an integratorto detect a number of drops per-unit-time entering the deflection tube.30. The article according to claim 24, wherein each deflection tubecomprises a metallic material.
 31. The article according to claim 24,wherein each collection tube comprises a plastic material.
 32. Thearticle according to claim 24, wherein the article comprises ten or moredeflection tubes and ten or more corresponding collection tubes.
 33. Thearticle according to claim 24, wherein the first opening of eachdeflection tube is aligned with the first opening of an adjacentdeflection tube such that the first openings are all collinear.
 34. Acell sorter system comprising an article according to claim
 24. 35. Thecell sorter system according to claim 34, further comprising a computerprocessor configured to identify when cells passed through a respectivedeflection tube are not in line with expectations.
 36. The cell sortersystem of according to claim 34, further comprising a motor toconfigured to move the article to a deactivated position.
 37. A methodcomprising introducing a sample into a cell sorter system according toclaim 34.